Over ten million solid phase immunoassays are run annually in the United States to detect the presence or absence of specific antibodies or antigens in biological samples, most commonly serum or urine. False positives in tests run in this manner are a common problem, and are sometimes so serious as to create a serious drawback to the use of the test. For example, recent efforts to screen blood samples for the presence of AIDS antibodies to certify blood available for transfusions have resulted in about 70% false positives on the initial screen (Medical World News (August 26, 1985) p. 15; Prentice, R. L. et al, Lancet (3 August 1985) p. 274-275). Furthermore, false positive results in screening for antibody to hepatitis B surface antigen threatens to undermine the usefulness of this test to screen patients for the costly hepatitis B vaccine (Annals of Internal Medicine (1985) 103:791-795). Elimination of false positives can generally be achieved through follow-up testing of positive samples using additional controls or different techniques. Although these approaches are expensive and time consuming, systematic internal noise control systems to eliminate false positives directly at the level of the initial assay have not been generally applied.
Some approaches to noise reduction associated with nonspecific binding of the test sample to the solid substrate in these assays have been applied, but these methods do not quantitatively account for the nonspecific binding. For example, Bullock, S. L. et al (J Infect Disease (1977) 136 (suppl): 279-285) disclose the use of albumin to block the solid substrate after the initial coating layer has been applied; Livesey, J. H. et al., Clin Chim Acta (1982) 123:193-198 and Hashida, S., et al, Clin Chim Acta (1983) 135:163-273 disclose the use of high concentrations of detergent, salt or protein in the sample buffer. These reagents are designed to interfere with nonspecific binding but cannot prevent it completely and cannot quantitate it so as to permit a precise accounting for its effect.
The problem of obtaining false positives is of course, aggravated when the substance to be detected in a biological fluid is present in quite low concentrations, thus necessitating the use of sample which is not greatly diluted. This increases the concentration of potentially interfering materials and thus increases the incidence of false positives. It is recognized by the invention herein, though not generally in the art, that an analogous circumstance arises in normal metabolism in situ at least for vertebrate systems, where specific receptors on target cells must be capable of preferentially binding their specific ligands and rejecting incorrect substances from the surroundings. Other workers have recognized that specific cell-cell adhesion requires a competition between nonspecific repulsion and specific binding (Bell, G. I., et al, Biophys J (1984) 45:1051-1064). It has also been demonstrated that cell surfaces in general bear a net negative charge (Mehrishi, J. N. in Progress in Biophysics and Molecular Biology (1972), vol 25, Butler, J. A., et al, eds, pp 3-69). It has also been recognized that the anionic surfaces of glomerular basement membrane and of blood vessel walls are responsible, in part, for inhibiting the transport of large anionic serum molecules across them (Seno, S., et al, Biorheology (1983) 20:653-662; Brenner, B. M., et al, Am J Physiol (1978) 234:5-6).
It has now been found that by mimicking to the appropriate degree (as determined by the method of the invention) the surface repulsion of cell surfaces by the initial layer in solid phase immunoassays, and by balancing the nonspecific binding or "noise" associated with the initial layer on a test portion and control surface portion of a solid support or supports, the specificity of solid phase immunoassay systems can be greatly improved and false positives minimized or eliminated.